Polyamine analogues as cytotoxic agents

ABSTRACT

Cytotoxic polyamine analogues are provided that are useful for treating diseases where it is desired to inhibit cell growth and/or proliferation, for example cancer and post-angioplasty injury.

RELATED APPLICATIONS

This application is related to U.S. patent application Ser. No.09/396,523, filed Oct. 15, 1999, which is hereby incorporated byreference as if fully set forth.

FIELD OF THE INVENTION

The invention in the field of chemistry and biochemistry relates to thesynthesis and use of novel polyamine analogue compounds withpharmacological or agricultural uses as cytotoxic agents. As drugs,these compounds are used to treat disorders of undesired cellproliferation, primarily cancer, alone or combined with other cytotoxicor anti-proliferative agents.

BACKGROUND OF THE INVENTION

Decades of research on the myriad of biological activities that thepolyamines, putrescine, spermidine and spermine play in cellularprocesses have shown the profound role they play in life (Cohen, S. S.,“A Guide to the Polyamines” 1998, Oxford University Press, New York). Aspolycations at physiological pH, they bind tightly to and stronglymodulate the biological activities of all of the anionic cellularcomponents. Specific and strong interactions have been associated withDNA and RNA together with their associated chromatin proteins (Tabor, H.et al. 1,4-Diaminobutane (putrescine), spermidine, and spermine. AnnRev. Biochem. 1976, 45, 285-306; Matthews, H. R. Polyamines, chromatinstructure and transcription. BioEssays, 1993, 15, 561-566). Specificinteractions of multicationic polyamines with microtubules has beenrecently shown (Wolff, J. Promotion of Microtubule Assembly byOligocations: Cooperativity between Charged Groups. Biochemistry, 1998,37, 10722-10729; Webb, H. K. et al.,1-(N-Allylamino)-11-(N-ethylamino)-4,8-diazaundecanes alter tubulinpolymerization. J Med. Chem. 1999 42(8):1415-21). Allosteric regulationof membrane-bound enzymes including acetylcholinesterase has been shown(Kossorotow, A. et al. Regulatory effects of polyamines onmembrane-bound acetylcholinesterase. Biochem. J. 1974, 144, 21-27).

There have also been reports on the involvement of polyamines in theinduction of apoptosis. Stefanelli and coworkers report that using HL60human leukemia cells (Stefanelli, C. et al. Spermine causes caspaseactivation in leukaemia cells. FEBS Letters, 1998, 437, 233-236) or acell-free model (Stefanelli, C. et al. Spermine triggers the activationof caspase-3 in a cell-free model of apoptosis. FEBS Letters, 1999, 451,95-98), addition of spermine led to the induction of apoptosis. Thisprocess was characterized by the release of cytochrome c frommitochondria, the dATP-dependent processing of pro-caspase-3 and theonset of caspase activity. This caspase activation was not blocked byantioxidants or inhibition of polyamine oxidase by MDL 72527. Thus theseworkers hypothesize a physiological role for the polyamines in thetransduction of a death message.

Due to its four positive charges at physiological pH, spermine ispredominantly bound to cellular components and its free concentration inthe cell is very low despite the high cellular content of this polyamine(Marton, L. J. et al. Polyamines as targets for therapeuticintervention. Annu. Rev. Pharmacol. Toxicol. 1995, 35, 55-91). Thus,spermine may have the characteristics of a damage-sensing molecule,since its free concentration may increase rapidly following insults tonucleic acids, membranes or other storage sites. This increase would beproportional to the extent of the damage and could transduce a deathsignal to the mitochondria.

Other workers have explored the toxic mechanisms of polyamines andpolyamine analogs. Poulin and coworkers showed that the deregulation ofpolyamine transport in L1210 cells over-expressing ornithinedecarboxylase (ODC) led to a lethal accumulation of spermidine (Poulin,R. et al. Induction of apoptosis by excessive polyamine accumulation inornithine decarboxylase-overproducing L1210 cells. Biochem. J 1995, 311,723-727). They showed that this lethal insult was due to the inductionof apoptosis. Polyamine oxidation was not responsible for the apoptosisobserved. Wallace and coworkers showed a similar non-oxidative lethalaction of spermine in BHK-21/C13 cells (Brunton, V. G. et al. Mechanismsof spermine toxicity in baby-hamster kidney (BHK) cells. Biochem. J.1991, 280, 193-198). They also showed that MDL 72527 exacerbated thetoxic effects of spermine.

Packham and Cleveland showed that the forced expression of ODC in 32D.3murine myeloid cells caused an apoptotic cell death following IL-3withdrawal (Packhani, G. et al. Ornithine decarboxylase is a mediator ofc-myc-induced apoptosis. Mol. Cell. Biol. 1994, 14, 5741-5747). ODCinduced cell death in a dose-dependent fashion, andα-difluoromethylornithine (DFMO), an irreversible inhibitor of ODCeffectively blocked ODC-induced cell death. Gerner and coworkers, in aseries of experiments with ODC over-expressing or polyamine transportregulation deficient cell lines, demonstrated that loss of feedbackregulation on the polyamine transport system is sufficient to induceapoptosis (Xie, X. et al. Loss of intracellular putrescine pool-sizeregulation induces apoptosis. Exp. Cell Res. 1997, 230, 386-392). Lossof regulation of the tight feedback controls on putrescine levels causedthe cells to undergo apoptosis in a putrescine dose-dependant manner.

Yanagawa and coworkers showed that the antiproliferative effects ofhepatocyte growth factor (HGF) involved the induction of apoptosis viaan increase in ODC activity with a resultant increase in intracellularpolyamine levels (Yanagawa, K. et al. The antiproliferative effect ofHGF on hepatoma cells involves induction of apoptosis with increase inintracellular polyamine concentration levels. Oncol. Rep. 1998, 5,185-190). Addition of the ODC inhibitor DFMO reduced the levels ofpolyamines and inhibited the apoptotic effects of HGF. This inhibitionof apoptotic effects was again reversed by the addition of exogenouspolyamines to the cells. The above reports indicate a clear apoptoticeffect upon loss of regulation of polyamine pool concentrations. It isalso clear that these effects occurred through a non-oxidativemechanism.

A series of modified spermine analogs, typified by N¹,N¹¹-diethylnorspermine (BE-3,3,3 also known as DENSPM), have been shownto super-induce the polyamine catabolic enzyme spermidine/spermineN¹-acetyltransferase (SSAT) and to work partially through an oxidativemechanism (Casero, R. A. et al. Spermidine/spermineN¹-acetyltransferase—the turning point in polyamine metabolism. FASEB J.1993, 7, 653-661). Porter and coworkers explored the cellular responsesto a series of these analogs and compared their cytotoxicity, inductionof SSAT and effects on the cell cycle (Kramer, D. L. et al. Effects ofnovel spermine analogues on cell cycle progression and apoptosis inMALME-3M human melanoma cells. Cancer Res. 1997, 57, 5521-5527). Theyconcluded that cytotoxicity could not be correlated with the level ofSSAT induction by these analogs, which left open the possibility thatadditional mechanism(s) could be involved. With only small changes inthe analog's structure, great variability was seen in the effects on thecell cycle.

Using related analogs, Hu and Pegg showed that the deregulated uptake ofpolyamine analogs by the polyamine transporter caused rapid induction ofapoptosis (Hu, R-H. et al. Rapid induction of apoptosis by deregulateduptake of polyamine analogues. Biochem. J. 1997, 328, 307-316).

Certain dibenzylputrescine analogs have been shown to haveanti-proliferative effects against human and rodent tumor cell lines.Frydman et al. describe the cytotoxicity against three squamous cellcarcinoma lines (SCC-38, SCC-4Y and SCC-13Y) and a rat hepatoma cellline (H-4-II-E) of the N¹, N₄-dibenzyl analogs of 1,3-diaminopropane,putrescine and cadaverine (Aizencang, G. et al. Antiproliferativeeffects of N¹, N⁴-dibenzylputrescine in human and rodent tumor cells.Cellular and Molecular Biology, 1998, 44 (4), 615-625 and U.S. Pat. No.5,677,350). IC₅₀ values of between 100 to 300 μM were found againstthese cell lines with the putrescine and cadaverine N¹, N⁴-dibenzylanalogs. These researchers describe the classic hallmarks of cellsundergoing apoptotic cell death: vacuole formation, decrease in size,changes in staining by trypan blue and adherence.

Frydman et al. also demonstrated that co-incubation with a specificpolyamine oxidase inhibitor, N¹, N⁴-bis(buta-2,3-dienyl)butanediamine(MDL 72527) caused a five-fold increase in the activity of the analogs.Although a moderate inhibition of [1,4-¹⁴C]-putrescine uptake was found(K_(iapp)=6.5+/−1.7 μM with N¹, N⁴-dibenzylputrescine compared toK_(mapp)=5.2+/−0.6 μM for putrescine), even a ten-fold excess ofputrescine over N¹, N⁴-dibenzylputrescine could not abolish its cellgrowth inhibitory effect. Moderate reductions in levels of intracellularpolyamines were measured after 72 h of drug treatment. These decreasesin the polyamine levels are of minor significance in comparison to thedecreases achieved with therapeutic approaches designed to depletepolyamines (see U.S. Pat. No. 6,172,261 B1).

Results from an in vivo antiproliferative study using N¹,N⁴-dibenzylputrescine (U.S. Pat. No. 5,677,350), suggested great promisefor these analogs. These studies showed significant reduction in theweights of the treated compared to control tumors. Nude mice aged fourweeks were subcutaneously inoculated with rat hepatoma H-4-II-E cells(10×10⁶ cells) or human melanoma II-B-Mel-J (5×10⁶ cells) and allowed todevelop for 15 to 24 days. Administration of 0.15% N¹,N⁴-dibenzylputrescine in the drinking water over 10 weeks showed notoxic effects on the animals. Several key observations were made inconjunction with these experiments. As stated above, the treatment withN¹, N⁴-dibenzylputrescine showed no liver or kidney damage following thetreatment. Metastatic lung tumors that were observed in the controlanimals did not appear in the treated animals. Most importantly, thegrowth of the tumors was strongly inhibited by a factor of 6 or 7-foldin the treated animals. Further study showed no significant changes inthe polyamine levels in the tumors from the treated in comparison to thecontrol animals.

A recent report suggests an explanation for the increased cytotoxicityobserved in the presence of MDL 72527 (Dai, H. et al. The polyamineoxidase inhibitor MDL-72,527 selectively induces apoptosis oftransformed hematopoietic cells through lysosomotropic effects. CancerResearch, 1999, 59, 4944-4954). This compound, previously reported to bea relatively non-toxic, selective polyamine oxidase (PAO) inhibitor, wasshown to induce apoptosis in transformed hematopoietic cells. It isinteresting to note that this compound was non-toxic to primary myeloidprogenitors. Cellular characterization of this compound revealedfeatures strikingly similar to those reported for the dibenzylputrescineanalogs above. Although this compound decreased the levels of putrescineand spermidine (it also increased the level of N¹-acetylspermidine),these effects were expected based on the compound's action as aninhibitor of PAO. The cytotoxic effects of this compound were notblocked by co-treatment with exogenous putrescine or spermidine. Theseeffects were also not influenced by over-expression or inhibition ofornithine decarboxylase (ODC), the rate-limiting polyamine biosyntheticenzyme. A well-characterized specific inhibitor of ODC, DFMO caused theincreased uptake of MDL 72527 leading to greater cytotoxicity buttreatment with putrescine/DFMO/MDL 72527 gave the same effects as MDL72527 alone.

In summary, these reports showed that N¹, N⁴-dibenzylputrescine andother similar analogs were not cytotoxic by depleting the intracellularpolyamine levels. The fact that a specific and potent PAO inhibitorincreased their activity suggested that a polyamine oxidase-mediatedmechanism was not responsible. Despite this limited knowledge about themechanism, these compounds did show moderate IC₅₀ values against severaldifferent cancer cell lines. They also showed the hallmarks of compoundsthat operate through an apoptotic mechanism. N¹, N⁴-dibenzylputrescineshowed significant promise in a mouse xenograft anti-tumor model. Thiscompound was orally active and showed no toxic effects even after a40-day treatment. Additional advantages of these compounds have beentheir easy and inexpensive synthesis.

Mitochondria apparently play a major role in apoptotic pathways. It isnow generally accepted that a decrease in the mitochondrial membranepotential is an early universal event of apoptosis (Mignotte, B. et al.Mitochondria and apoptosis. Eur. J Biochem. 1998, 252, 1-15).Mitochondria participate in the early steps of apoptosis, in response tomany stimuli, through the release of cytochrome c into the cytoplasm.Recent literature reports indicate that many molecules, includingseveral clinically promising agents, induce apoptosis through therelease of cytochrome c from the mitochondria. One well-establishedmechanism for this release is the swelling of the mitochondrial innermembrane followed by rupture of the outer membrane/matrix. The releaseof the positively charged cytochrome c protein from the mitochondria isstrongly linked to the induction of apoptosis (Green, D. R. et al.Mitochondria and Apoptosis. Science, 1998, 281, 1309-1312). The releasedcytochrome c initiates a complex pathway that ultimately results in theactivation of caspase-3.

Tamanoi and coworkers showed that a set of four structurally diversefarnesyltransferase inhibitors induce the release of cytochrome c frommitochondria of ν-K-ras-transformed normal rat kidney cells (Suzuki, N.et al. Farnesyltransferase inhibitors induce cytochrome c release andcaspase 3 activation preferentially in transformed cells. Proc. Natl.Acad. Sci. USA, 1998, 95, 15356-115361). They showed that this releaseresulted in caspase-3 activation and was observed preferentially intransformed cells compared to the normal cells.

Debatin and coworkers showed that betulinic acid, a melanoma-specificcytotoxic agent, triggered CD95 (APO-1/Fas)- and p53-independentapoptosis via release of cytochrome c and apoptosis inducing factor(AIF) from the mitochondria into the cytosol (Fulda, S. et al.Activation of mitochondria and release of mitochondrial apoptogenicfactors by betulinic acid. J. Biol. Chem. 1998, 273, 33942-33948). Thefact that this drug-induced apoptosis (via a direct effect onmitochondria) did not involve two common resistance mechanisms suggeststhat betulinic acid may bypass some forms of drug resistance (Fulda, S.et al. Betulinic acid triggers CD95 (APO-1/Fas)- and p53-independentapoptosis via activation of caspases in neuroectodermal tumors. CancerRes. 1997, 57, 4956-4964).

An additional agent, presently in Phase III trials of metastatic breastand ovarian cancer, lonidamine(1-[(2,4-dichlorophenyl)methyl]-1H-indazole-3-carboxylic acid), alsoacts independently of p53 status via a direct action on themitochondrial permeability transition pore (Ravagnan, L. et al.Lonidamine triggers apoptosis via a direct, Bcl-2-inhibited effect onthe mitochondrial permeability transition pore. Oncogene 1999, 18,2537-2546).

The early universal apoptotic event of a decrease in the mitochondrialmembrane potential may occur by the opening of pores in the innermembrane of mitochondria. These pores allow the passage of compounds ofmolecular weight of <1500 Da through the membrane and several of thesehave been directly linked to the induction of apoptosis.

Citation of the above documents is not intended as an admission that anyof the foregoing is pertinent prior art. All statements as to the dateor representation as to the contents of these documents is based on theinformation available to the applicant and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

SUMMARY OF THE INVENTION

The present invention relates to the synthesis and growth inhibitoryproperties of polyamine analogues and their use as drugs, asagricultural or as environmentally useful agents. Preferably, theanalogues are derivatives of spermine, spermidine and putrescine, suchas derivatives of dibenzylputrescine.

The analogues of the present invention include derivatives of spermine,spermidine and putrescine, as well as analogs thereof, substituted atone or both of their terminal (alpha, or α, and omega, or ω) nitrogenatom positions. Preferred analogues are substituted at both positions.The substitutions may be with the same or different chemical moieties.Moreover, the analogues may be substituted at one or more internalnitrogen and/or carbon positions along the polyamine backbone by a lowmolecular weight chemical moiety.

A preferred embodiment is a highly cytotoxic analogue withpharmaceutical utility as an anti-cancer chemotherapeutic. Such ananalogue would have an IC₅₀ in the micromolar or submicromolar rangeagainst tumor cells. Preferred compounds with such activity includecompounds 1313 and 1327 as described herein. Additional preferredcompounds include spermine analogues substituted at both terminalnitrogen atoms by identical substituents.

Preferred substituents are structures that increase cytotoxicity orotherwise enhance the inhibition of cell growth, proliferation,metastases, or neoplasm. Such additional substituents include theaziridine group and various other aliphatic, aromatic, mixedaliphatic-aromatic, or heterocyclic multi-ring structures.

More specifically, a polyamine analogue or derivative of the inventionincludes one that is cytotoxic and has the formula:R₁—X—R₂wherein

-   -   R₁ and R₂ are independently H or a moiety selected from the        group consisting of a straight or branched C₁₋₁₀ aliphatic,        alicyclic, single or multi-ring aromatic, single or multi-ring        aryl substituted aliphatic, aliphatic-substituted single or        multi-ring aromatic, a single or multi-ring heterocyclic, a        single or multi-ring heterocyclic-substituted aliphatic and an        aliphatic-substituted aromatic, and halogenated forms thereof;        and    -   X is a polyamine with two terminal amino groups, —(CH₂)₃—NH—, or        —CH₂—Ph—CH₂—.

Preferably, the polyamine is linear or selected from spermine,spermidine, or putrescine. R₁ and R₂ may be identical or different andare preferably not simultaneously unsubstituted benzyl moieties.Halogenated moieties include those substituted with fluorine, chlorine,bromine, and iodine.

Alternatively, X is —(CH₂)₃—NH— or —CH₂—Ph—CH₂—, where “Ph” represents aphenyl moiety, and R₁ and R₂ are as described above.

Disubstituted polyamine analogues, preferably also containing a reportergroup, may also be employed as assay or biochemical probes.

Once a cytotoxic polyamine analogue has been identified, it can readilybe further optimized by structural and functional comparisons with otherpolyamine analogues to improve its utility. Examples of suchimprovements include, but are not limited to, increased cytotoxicity,enhanced metabolic stability, enhanced specificity, ease of handling andadministration, non-incorporation into cellular polyamine pools, anddecreases in side effects.

The present invention is also directed to compositions comprising apolyamine analog. Preferably, the composition is a pharmaceuticalformulation useful for treating a disease or condition in which theinhibition of cell growth or proliferation is desirable, comprising acomposition as described above and a pharmaceutically acceptableexcipient. The pharmaceutical composition may further include additionalcytotoxic compounds or an inhibitor of polyamine synthesis, such asDFMO. Other combinations include the above pharmaceutical compositionand one or more additional agents otherwise known to be useful fortreating said disease or condition

This invention also provides a method for inhibiting cell growth orproliferation comprising contacting the cell(s) with an analogue of theinvention. Such methods include treating a disease or a condition in asubject associated with undesired cell proliferation by administering tosaid subject an effective amount of a pharmaceutical composition asdescribed above. The undesired cell proliferation may be associated withproliferation of cells of the immune system, cells of the vascularneontima, tumor cells or with undesired angiogenesis. Preferred diseasesto be treated as above include cancer or post-angioplasty injury.

Thus the analogues of the invention, alone or in combination with otheragents, may be used for the treatment of cancer and other diseases ofunwanted cellular proliferation, including angiogenesis and post-injurycell growth. Preferably, such treatments act by inhibiting cell growthor by the induction of apoptosis. As such, they may act by cytostaticand/or cytotoxic mechanisms. The analogues of the invention,individually or in combinations with or without other agents, may alsobe used to treat hypertension, osteoporosis, Alzheimer's disease,ischemia, autoimmune diseases, psychosis, depression, strokes,cardiovascular disease, allergies, asthma, tissue rejection duringtransplantation, infection with microorganisms or parasites, as well asplant pathogens including fungi. The analogues of the invention may alsobe efficacious as anti-diarrheal, anti-peristaltic, anti-spasmodic,anti-viral, anti-psoratic and insecticidal agents.

The present invention is also directed to a series of polyamineanalogues useful in diagnostic compositions. Methods for the synthesisof such compounds are also described.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a reaction scheme for the production of polyamine analogswithin the scope of the invention.

FIG. 1B shows the synthesis of mono- and unsymmetrically disubstitutedanalogs.

FIG. 2 shows the structures of polyamine analogues Ori 1313 and Ori1327.

FIG. 3 is a table containing preferred polyamine analogues of theinvention where the general structure of the analogues is shown at thetop of the table. Included are analogues (or “Ori”) 1313 and 1327. Allstructures shown are derived from putrescine (1,4-diaminobutane) unlessotherwise noted.

FIG. 4 is a table containing additional polyamine analogues of theinvention.

FIGS. 5A and 5B show cytotoxicity of ORI 1313 (-▪-) and ORI 1327 (-♦-)against tumor cell lines (see Example 1 herein).

FIGS. 6A and 6B show time courses of ORI 1313 and OR 1327 cytotoxicity(see Example II herein).

FIGS. 7A-7C show induction of apoptosis by polyamine analogues OR 1313and ORI 1327.

FIGS. 8A and 8B show cytotoxicity for tumor cells expressing MDR-1 bypolyamine analogues ORI 1313 and ORI 1327.

FIG. 9 shows that the polyamine analogues ORI 1313 and ORI 1327 do notalter cellular polyamine levels.

FIG. 10 shows that cytotoxicity of polyamine analogue ORI 1313 involvescaspase-3 (see Example VI herein).

FIG. 11 is a table containing polyamine analogues, including halogenatedanalogues, of the invention.

FIG. 12 shows additional symmetrical polyamine analogues of theinvention, including halogenated analogues.

FIG. 13 is a graph illustrating inhibition of tumor growth in ORI 1313treated A375 human melanoma xenografts in mice (see Example IX herein).

DETAILED DESCRIPTION

The present inventor has designed novel polyamine analogue compoundsdisplaying both in vivo and in vitro cytotoxicity. Such compounds areuseful as drugs in a number of diseases, particularly cancer. They canalso be used as a component of novel drug combinations with, forexample, a polyamine synthesis inhibitor such as DFMO (which inhibitsornithine decarboxylase) or with other cytotoxic agents. A compound ofthe present invention is generally useful in diseases or conditions inwhich inhibition of cell growth is desirable, and also has agriculturaland environmental uses based on its cytotoxicity.

The inventor found that various chemical groups can be attached to apolyamine to give it advantageous properties as an inhibitor of cellgrowth and/or proliferation.

In a preferred aspect of the invention, the analogues are advantageousin the treatment of human melanoma. Human melanoma is a growing healthproblem in the United States and much of the world. Increased solarradiation exposure due to ozone depletion makes this disease a profoundhealth concern for aging populations and for future generations.Malignant melanoma is considered to be a chemotherapy-refractory tumorand commonly used anticancer drugs do not appear to modify the prognosisof metastatic disease (Serrone, L. et al. The chemoresistance of humanmalignant melanoma: an update. Melanoma Res. 1999, 9, 51-58). Preferredembodiments of the polyamine analogues of the invention have been foundto display dramatic selectivity toward melanoma cell lines.

Definitions

As used herein, the term “polyamine” includes naturally occurringpolyamines, such as putrescine, spermine or spermidine, as well as othernaturally occurring polyamines, such as caldopentamine,homocaldopentamine, N⁴-bis(aminopropyl)norspermidine, thermopentamine,N⁴-bis(aminopropyl)spermidine, caldohexamine, homothermohexamine andhomocaldohexamine, cadaverine, aminopropylcadaverine, homospermidine,caldine (norspermidine), 7-hydroxyspermidine, thermine (norspermine),thermospermine, canavalmine, aminopropylhomospermidine, andaminopentylnorspermidine.

The term also embraces longer linear polyamines, branched polyamines,and the like, which may have between 2 and about 10 nitrogen atoms. Thenitrogen atoms are generally separated by 2 to 6 carbon atoms along thelinear chain. Also included in this definition are polyamine derivativesor analogues comprising a basic polyamine chain with any of a number offunctional groups covalently bonded to a C atom or a terminal orinternal N atom. These include N¹-monosubstituted polyamine analogues,as well as substitution of carbon atoms α to secondary nitrogens andacylation of nitrogens to slow degradation by polyamine oxidase. Theselective primary mono-substitution of polyamines is known (Krapcho, A.P. et al. Mono-protected diamines.N-tert-butoxylcarbonyl-α,ω-alkanediamines from α,ω-alkanediamines. Syn.Comm. 1990, 20, 2559-2564; Blagbrough, I. S. et al. Practical Synthesisof unsymmetrical polyamine amides. Tetrahedron Lett. 1998, 39, 439-442).Alternatively, methyl groups can be introduced α to the terminal aminogroups of spermine (Lakanen, J. R. et al., J. Med. Chem. 35:724-734,1992).

Various polyamine analogues alkylated at internal carbons can also bereadily synthesized. 5-carboxyspermine, tetra tBoc-5-carboxyspermine andits acid chloride are synthesized according to Huber, H. et al., J.Biol. Chem. 271:27556-27563, 1994. The resulting acid chloride can thenbe reacted with various nucleophilic reagents to producecarboxy-substituted polyamine analogues following removal of the tBocgroup. Alternatively, the carboxy intermediate can be reduced to anintermediate that is used to synthesize numerous additional analogues.

A “reporter moiety” is a chemical moiety forming part of a probe whichrenders the probe detectable (either directly or, for example, throughenzymatic enhancement) and hence permits the localization of the probe.A reporter is detectable either because it itself emits a detectablesignal, or by virtue of its affinity for a reporter-specific partnerwhich is detectable or becomes so by binding to, or otherwise reactingwith, the reporter.

The various polyamine analogue compounds disclosed herein are identifiedby an identifier number scheme (using four digit compound numbers aloneor in combination with an “ORI”, or “Ori” identifier). Irrespective ofwhat identifying scheme is used, the identifier merely represents theactual molecular structure of the compound involved and imposes nolimitation on said compound.

Polyamine Analogue Structure and Synthesis

The polyamine analogues of the present invention are generallysubstituted or derivatized forms of existing or novel polyamines.Preferably, the analogues are derivatives of spermine, spermidine andputrescine. More preferably, the analogues are substituted at least atone or both of the terminal (alpha, or α, and omega, ω) nitrogen atompositions of an underlying polyamine. The analogues are preferablysubstituted at both positions. Most preferred are analogues with an IC₅₀against tumor cells in the micromolar range (including from 1 to about600, 1 to about 300, 1 to about 200, 1 to about 100, 1 to about 50, 1 toabout 20, 1 to about 15, 1 to about 10, 1 to about 9, 1 to about 8, 1 toabout 7, 1 to about 6, 1 to about 5, 1 to about 4, 1 to about 3, and 1to about 2 μM) and submicromolar range (including from about 0.01 to 1,about 0.1 to 1, about 0.2 to 1, about 0.3 to 1, about 0.4 to 1, about0.5 to 1, about 0.6 to 1, about 0.7 to 1, about 0.8 to 1, and about 0.9to 1 μM).

FIG. 1A shows an exemplary reaction Scheme I for the production ofsubstituted polyamines that have 3, 4 or 5 carbon atoms separating twoterminal amino groups in a linear chain. This reaction is an extremelydirect synthetic method and may be conducted in a single reactionvessel. The exemplary polyamine reactant with 4 carbon atoms isputrescine. The product of the reaction using putrescine isN¹,N⁴-dibenzylputrescine. Following the reaction, the polyamineanalogues are readily purified by column chromatography orcrystallization.

Similar reactions can be conducted with spermine, spermidine and otherpolyamines to produce analogues of the invention. Such additionalreactions are known in the art and may include appropriate steps toprotect functional groups within the structures of larger polyaminessuch as spermine and spermidine.

The reaction scheme in FIG. 1A is readily modified to produce cytotoxicpolyamine analogues of the invention by the use of aldehydes other thanthe exemplary aromatic aldehyde indicated. Thus reactions with aldehydescontaining cyclic or aliphatic moieties, as well as substituted formsthereof may be used to produce additional analogues of the invention.Cyclic moieties may of course be either homocyclic or heterocyclic, aswell as aromatic or aryl, to permit production of the analogues of theinvention. The aliphatic moieties may of course contain one or morenon-carbon heteroatoms.

Examples of cyclic moieties include multi-ring and multi-single-ringgroups as well as the bonds or straight chain groups that attachdifferent ring structures in a multiple ring head group. Examples ofsuch groups for covalent attachment of a ring structure are amide,sulfonamide, ether, thioether, ester, —C—C— and —C—N— and —N—N— bonds.The ring structures can also be individually substituted.

Examples of heterocyclic moieties include, but are not limited to,pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, biphenyl, furanyl,pyrrolyl, 1,2-diazolyl, imidazolyl, 1H,1,2,3-triazolyl,1H-1,2,3,4-tetrazolyl, thiazolyl, oxazolyl, 1,3,4-thiadiazolyl,pyridinyl, pyrimidyl, 1,2-diazinyl, 1,4-diazinyl, 1,3,5-trizinyl,dibenzofuranyl, acridinyl, 2,1,3-benzothiadiazole, isoquinolinyl,quinolinyl, benzufuranyl, isobenzofuranyl, 1,3-benzodiazinyl,phenazinyl, phenoxazinyl, phenothiazinyl, pyran, chromenyl, xanthenyl,indolizinyl, isoindolyl, indolyl, purinyl, phthalazinyl, naphthyridinyl,quinoxalinyl, quinazolinyl, cinnolinyl, ptericinyl, carbazolyl,β-carbolinyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl,isothiazoly, furazanyl, indolinyl, isoindolinyl, quinuclidinyl, andbiotinyl.

Examples of aromatic moieties include, but are not limited to, phenylnaphthyl, 1-, 2-, or 3-biphenyl, indenyl, acenaphthylenyl, anthracenyl,phenanthrenyl, phenalenyl, triphenylenyl pyrenyl, anddiphenylmethylenyl.

Examples of aliphatic moieties include, but are not limited to,straight-chain, branched and cyclic hydrocarbons; C₂₋₁₀ alkanes; C₃₋₁₀alkenes containing 1 to 3 unsaturations; C₃₋₁₀ alkynes containing 1 to 3unsaturations; branched C₃₋₁₀ alkanes, alkenes and alkynes; polycyclicaliphatic hydrocarbons and steroid-like ring systems that include C₃₋₈cycloalkyl, adamantyl, camphoryl, and cholesteryl.

Moreover, the polyamine reactants used in reactions similar to thatexemplified in FIG. 1A may be derivatized at one or more internalnitrogen and/or carbon positions along the linear backbone by a lowmolecular weight chemical moiety. Examples of such moieties are found inthe following list:

halogen cyclohexyl ethoxyl propyl ester methyl cycloheptyl propoxylisopropyl ester ethyl cyclooctyl thio cyano propyl cyclononyl methylthioisocyanato isopropyl cyclodecyl ethylthio trifluoromethyl butyl hexylpropylthio trichloromethyl isobutyl 2-hexyl butylthio tribromomethyltert-butyl 3-hexyl isopropylthio azido pentyl allyl nitro Acetoxy2-pentyl vinyl amino Carboxamide 3-pentyl acetylenic acetamideN-methylcarbox- amide neopentyl propargylic formamide N,N-dimethyl-carboxamide cyclopentyl homopropargylic carboxylic N-ethylcarboxamidecyclopropyl hydroxyl methyl ester N,N-diethylcarbox- amide cyclobutylmethoxyl ethyl ester

Mono and multi-substituted forms of the moieties are also encompassed bythe invention.

Preferred Polyamine Analogues and Derivatives

A preferred embodiment is a highly cytotoxic analogue withpharmaceutical utility as an anti-cancer chemotherapeutic. Preferredanalogues with such activity include compounds 1313 and 1327 which havethe structures shown in FIG. 2. These analogues are more potent thanN¹,N⁴-dibenzylputrescine, are cytotoxic to tumor cells at low micromolarconcentrations, appear to induce apoptosis, and demonstrate efficacy inas little as one hour of treatment. Moreover, the analogues appear toinduce apoptosis in tumor cell lines and are unexpectedly able toinhibit growth of tumor cell lines that express the multi-drugresistance. Furthermore, these analogues are also of particularspecificity and efficacy for inhibiting cell growth and proliferation inmelanoma cells.

Additional preferred compounds of the invention are putrescine analogueshaving the indicated R Groups as shown in FIG. 3 with the exception ofanalogues “1191” and “1192”. The analogues listed by number in FIG. 3are derivatives of putrescine whereby each indicated R Group is presenton both terminal nitrogen atom positions. The indicated IC₅₀ values arefor the human breast tumor cell line MDA-MB-231 (“MDA”) and the humanPC-3 prostate tumor cell line.

Further analogues of the invention include analogues of1,3-diaminopropane, spermine, spermidine, and other polyaminesderivatized with the R Groups of FIG. 3. For all linear polyamineanalogues, including putrescine analogues, of the invention containingsuch R Groups, the derivatization need not be at both ends of thepolyamine backbone, but may instead be at only one end.

Additional analogues of the invention are shown in FIG. 4. It is readilyapparent that the analogues are all of the formula R₁—X—R₂ as presentedabove. Thus each “R₁” and “R₂” group of FIG. 4 may be independentlyconsidered a moiety for the derivatization of any polyamine, including,but not limited to, 1,3-diaminopropane, putrescine, spermidine, andspermine.

Other preferred compounds of the invention are shown in FIGS. 11 and 12.Of these, it is relevant to note that compounds 1441 and 1436 do notcontain a linear polyamine as the core. Also, compound 1429 containsfurther substituents on internal carbon atoms of the polyamine.Compounds 1368, 1367, 1366, 1365, 1364, and 1363 comprise —(CH₂)₃—NH— asthe core. They may also be viewed as asymmetrical polyamine analogues.In particular, compounds 1368 and 1367 may also be used as probes intothe mechanism of action of cytotoxic polyamine analogue compounds. Otherasymmetric polyamine analogues include compounds 1318, 1317, 1316, 1310,1303, 1302, and 1301.

Preferred analogue compounds of the invention include derivatives of thecompounds presented in FIGS. 3, 4, 11 and 12 as well as those withpharmaceutical utility as an anti-cancer, anti-viral, anti-microbial,anti-bacterial, anti-parasitic or anti-fungal chemotherapeutic basedupon their cytotoxic properties.

The further derivatization or optimization of compounds having adesirable activity may be achieved by structural and functionalcomparisons with other polyamine analogues and derivatives of theinvention to incorporate particular structural elements of otheranalogues into the compound being optimized. The structural elementswill be selected based on the expectation of improving functionalitiessuch as, but not limited to, cytotoxicity, metabolic stability,specificity, handling and administration, binding affinity,non-incorporation into cellular polyamine pools, and decreases in sideeffects.

The resultant compounds modified by the introduction of such structuralelements may be of any structure, including those within the limits ofthe polyamine analogues and derivative structures defined herein. Stateddifferently, the resultant compounds may have one or more additionalatoms or functional groups and/or removal of one or more atoms orfunctional groups after optimization, resulting in a compound eitherwithin or beyond the limits of the polyamine analogues and derivativestructures defined herein.

Multiple iterations of optimizing compounds with preferred activity maybe conducted to further improve the polyamine analogue.

Analytical and Diagnostic Uses

The polyamine analogues and derivatives of the invention may also beused as reporter molecules and probes to assay their localization withcellular factors which may be other pharmacological targets. Suchfactors include membranes, soluble and insoluble proteins, and nucleicacids.

Pharmaceutical and Therapeutic Compositions and Applications

The polyamine analogues and derivatives of the invention, as well as thepharmaceutically acceptable salts thereof, may be formulated intopharmaceutical compositions. Pharmaceutically acceptable acid salts ofthe compounds of the invention which contain basic groups are formedwhere appropriate with strong or moderately strong, non-toxic, organicor inorganic acids in the presence of the basic amine by methods knownin the art. Exemplary of the acid salts that are included in thisinvention are maleate, fumarate, lactate, oxalate, methanesulfonate,ethanesulfonate, benzenesulfonate, tartrate, citrate, hydrochloride,hydrobromide, sulfate, phosphate and nitrate salts. Additionalillustrative acids which form suitable salts include hydrochloric,hydrobromic, sulfuric, and phosphoric acids; acetic, glycolic, lactic,pyruvic, malonic, succinic, glutaric, .alpha.-ketoglutaric,.alpha.-ketocaproic, .alpha.-ketoisocaproic, .alpha.-ketoisovaleric,fumaric, malic, tartaric, citric, ascorbic, maleic, hydroxymaleic,benzoic, hydroxybenzoic, phenylacetic, cinnimic, salicylic, and2-phenoxybenzoic acids; and sulfonic acids such as methane sulfonic acidand 2-hydroxyethane sulfonic acid. Acid metal salts such as sodiummonohydrogen orthophosphate and potassium hydrogen sulfate are alsoencompassed. As stated above, the compounds of the invention possesscytotoxic properties that are exploited in the treatment of any of anumber of diseases or conditions, most notably cancer. A composition ofthis invention may be active per se, or may act as a “pro-drug” that isconverted in vivo to active form.

The compounds of the invention, as well as the pharmaceuticallyacceptable salts thereof, may be incorporated into convenient dosageforms, such as capsules, impregnated wafers, tablets or injectablepreparations. Solid or liquid pharmaceutically acceptable carriers maybe employed. Pharmaceutical compositions designed for timed release mayalso be formulated.

Optionally, the compositions contain anti-oxidants, surfactants and/orglycerides. Examples of anti-oxidants include, but not limited to, BHT,vitamin E and/or C. Examples of glycerides include, but are not limitedto, one or more selected from acetylated or unsubstitutedmonoglycerides; medium chain triglycerides, such as those found in oils;and caprylocaproyl macrogol-8 glycerides.

Preferably, the compounds of the invention are administeredsystemically, e.g., by injection or oral administration. When used,injection may be by any known route, preferably intravenous,subcutaneous, intramuscular, intracranial or intraperitoneal.Injectables can be prepared in conventional forms, either as solutionsor suspensions, solid forms suitable for solution or suspension inliquid prior to injection, or as emulsions.

Solid carriers include starch, lactose, calcium sulfate dihydrate, terraalba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearateand stearic acid. Liquid carriers include syrup, peanut oil, olive oil,saline, water, dextrose, glycerol and the like. Similarly, the carrieror diluent may include any prolonged release material, such as glycerylmonostearate or glyceryl distearate, alone or with a wax. When a liquidcarrier is used, the preparation may be in the form of a syrup, elixir,emulsion, soft gelatin capsule, liquid containing capsule, sterileinjectable liquid (e.g., a solution), such as an ampule, or an aqueousor nonaqueous liquid suspension. A summary of such pharmaceuticalcompositions may be found, for example, in Remington's PharmaceuticalSciences, Mack Publishing Company, Easton Pa. (Gennaro 18th ed. 1990).

The pharmaceutical preparations are made following conventionaltechniques of pharmaceutical chemistry involving such steps as mixing,granulating and compressing, when necessary for tablet forms, or mixing,filling and dissolving the ingredients, as appropriate, to give thedesired products for oral or parenteral administration. Otherpreparations for topical, transdermal, intravaginal, intranasal,intrabronchial, intracranial, intraocular, intraaural and rectaladministration may also be prepared. The pharmaceutical compositions mayalso contain minor amounts of nontoxic auxiliary substances such aswetting or emulsifying agents, pH buffering agents and so forth.

Although the preferred routes of administration are systemic, thepharmaceutical composition may be administered topically ortransdermally, e.g., as an ointment, cream or gel; orally; rectally;e.g., as a suppository, parenterally, by injection or continuously byinfusion; intravaginally; intranasally; intrabronchially;intracranially; intraaurally; or intraocularly.

For topical application, the compound may be incorporated into topicallyapplied vehicles such as a salve or ointment. The carrier for the activeingredient may be either in sprayable or nonsprayable form.Non-sprayable forms can be semi-solid or solid forms comprising acarrier indigenous to topical application and having a dynamic viscositypreferably greater than that of water. Suitable formulations include,but are not limited to, solution, suspensions, emulsions, creams,ointments, powders, liniments, salves, and the like. If desired, thesemay be sterilized or mixed with auxiliary agents, e.g., preservatives,stabilizers, wetting agents, buffers, or salts for influencing osmoticpressure and the like. Preferred vehicles for non-sprayable topicalpreparations include ointment bases, e.g., polyethylene glycol-1000(PEG-1000); conventional creams; gels; as well as petroleum jelly andthe like.

Also suitable for topical application are sprayable aerosol preparationswherein the compound, preferably in combination with a solid or liquidinert carrier material, is packaged in a squeeze bottle or in admixturewith a pressurized volatile, normally gaseous propellant. The aerosolpreparations can contain solvents, buffers, surfactants, perfumes,and/or antioxidants in addition to the compounds of the invention.

For the preferred topical applications, especially for humans, it ispreferred to administer an effective amount of the compound to a targetarea, e.g., skin surface, mucous membrane, eyes, etc. This amount willgenerally range from about 0.001 mg to about 1 g per application,depending upon the area to be treated, the severity of the symptoms, andthe nature of the topical vehicle employed.

The compositions of the invention be given in combination with one ormore additional compounds that are used to treat the disease orcondition. For treating cancer, the polyamine analogues and derivativesmay be given in combination with anti-tumor agents, such as mitoticinhibitors, e.g., vinblastine; alkylating agents, e.g.,cyclophosphamide; folate inhibitors, e.g., methotrexate, pritrexim ortrimetrexate; antimetabolites, e.g., 5-fluorouracil and cytosinearabinoside; intercalating antibiotics, e.g., adriamycin and bleomycin;enzymes or enzyme inhibitors, e.g., asparaginase; topoisomeraseinhibitors, e.g., etoposide; or biological response modifiers, e.g.,interferon. In fact, pharmaceutical compositions comprising any knowncancer therapeutic in combination with the polyamine analogues andderivatives disclosed herein are within the scope of this invention. Thepresent compounds may also be administered in combination with apolyamine synthesis inhibitor such as DFMO.

The pharmaceutical compositions of the invention may also comprise oneor more other medicaments such as anti-infectives includingantibacterial, anti-fungal, anti-parasitic, anti-viral, andanti-coccidial agents.

Typical single dosages of the compounds of this invention are betweenabout 1 ng and about 1 g/kg body weight. The dose is preferably betweenabout 0.01 mg and about 500 mg/kg body wt. and, most preferably, betweenabout 0.1 mg and about 100 mg/kg body wt. For topical administration,dosages in the range of about 0.01-20% concentration of the compound,preferably 1-5%, are suggested. A total daily dosage in the range ofabout 1-200 mg is preferred for oral administration. The foregoingranges are, however, suggestive, as the number of variables in regard toan individual treatment regime is large, and considerable excursionsfrom these recommended values are expected and may be routinely made bythose skilled in the art.

Effective amounts or doses of the compound for treating a disease orcondition can be determined using recognized in vitro systems or in vivoanimal models for the particular disease or condition. In the case ofcancer, many art-recognized models are known and are representative of abroad spectrum of human tumors. The compounds may be tested forinhibition of tumor cell growth in culture using standard assays withany of a multitude of tumor cell lines of human or nonhuman animalorigin. Many of these approaches, including animal models, are describedin detail in Geran, R. I. et al., “Protocols for Screening ChemicalAgents and Natural Products Against Animal Tumors and Other BiologicalSystems (Third Edition)”, Canc. Chemother. Reports, Part 3, 3:1-112.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

EXAMPLE I Cytotoxicity of Putrescine Analogs

ORI 1313 and ORI 1327 displayed significant cytotoxic activity againstSK-MEL-5 and MDA cell lines (see FIGS. 5A and 5B, ORI 1327, -♦-; ORI1313, -▪-). Briefly, cells were plated at 1500 (SK-MEL-5) or 5000 (MDA)cells/well in a 96-well plate and allowed to adhere for 1 day. Theindicated analogues were added and the cells were incubated for 3 days,when cell growth was evaluated by MTS assay (Promega). Vertical axisrepresents cell number as a % of no drug treatment controls.Additionally, significant activity was seen against PC-3 cells (seeFIGS. 4 and 11).

These two agents represent significant improvements in potency over N¹,N⁴-dibenzylputrescine. IC₅₀ values against SK-MEL-5 cells weredetermined to be 4.1 μM and 0.71 μM for ORI 1313 and ORI 1327,respectively. The data shows ORI 1313 and ORI 1327 to have IC₅₀ valuesof 12 μM and 0.65 μM against MDA breast carcinoma cells, respectively.N¹, N⁴-dibenzylputrescine showed an IC₅₀ of 192 μM when tested againstthe MDA cells. Thus ORI 1313 and ORI 1327 showed a 16-fold or 295-foldincrease, respectively, over N¹, N⁴-dibenzylputrescine in potency ofcytotoxic activity against MDA cells.

N¹, N³-dibenzyl analog of 1,3-diaminopropane has also been tested anddetermined to have an IC₅₀ value of 28.8 μM against MDA cells.

EXAMPLE II Time Course of Polyamine Analogue Cytotoxicity in Tumor Cells

Even after only one hour of treatment, polyamine analogs displayedsignificant cytotoxicity in MDA cells (see FIGS. 6A and 6B). Briefly,MDA-MB-231 cells were plated at 5000 cells/well in a 96-well plate andallowed to adhere for 1 day. The indicated analogues were added and thecells were incubated for the various times shown. The cells were thenwashed and fresh analogue-free media was added. After a total of 3-days,cell growth was evaluated by MTS assay (Promega). Vertical axisrepresents cell number as a % of no drug treatment controls

These findings suggest that the polyamine analogues can inducecytotoxicity without need for prolonged depletion of polyamines and thatremoval of the drug from the medium does not enable the cells to recoveror regrow. This observation may have significant implications in theclinical use of these agents. Given a sufficiently high selectivity forcancer versus normal cells, contact with a threshold concentration ofthe analogue for a relatively short period will induce tumor cell deathwithout need for maintenance of analogue concentration. Thus high,sustained levels of analogue or prolonged treatments may not be requiredfor effective anti-tumor therapy with these compounds.

EXAMPLE III Cytotoxic Polyamine Analogues Induce Apoptosis

Cell cycle analysis was performed using fluorescent activated cellsorter (FACS) analysis on polyamine analogue treated MDA cells (seeFIGS. 7A-7C). Briefly, MDA-MB-231 cells were grown in the presence ofORI 1313 or ORI 1327 for various times then fixed with ethanol andstained with propidium iodide. Cells were analyzed by FACS, with theareas on the histograms designating DNA content and cell cycle stage:M1, G1; M2, S; M3, G2/M; M4, <2N (apoptotic cells). FIG. 7A shows theresults with untreated cells; 7B shows the results with 32 μM ORI 1313treatment for 48 hours; and 7C shows the results with 3 μM ORI 1327treatment for 11 hours.

This analysis showed a high apoptotic fraction after ORI 1313 treatment(32 μM, 11 hr treatment gave 21%, 24 hr treatment gave 49% and 48 hrtreatment gave 30% <2N DNA content) and showed moderate apoptotic cellsafter ORI 1327 treatment (3 μM, 11 hr treatment gave 15% <2N DNAcontent). These data show that the analogues potently induce the celldeath apparatus of the tumor cells

EXAMPLE IV Cytotoxic Polyamine Analogues are Cytotoxic for MDR TumorCells

An unexpected observation made during the cellular characterization ofORI 1313 and ORI 1327 was their growth inhibitory activity against themulti-drug resistant (MDR) uterine sarcoma cell line, MES-SA/Dx5 (seeFIGS. 8A and 8B). Briefly, cells were plated at 1000 cells/well in a96-well plate and allowed to adhere for 1 day. The indicated analoguewas added and the cells were incubated for 3 days when cell growth wasevaluated by MTS assay (Promega). Vertical axis represents cell numberas a % of no drug treatment controls

The MES-SA/Dx5 cell line was developed by selection on doxorubicin andover-expresses the mRNA of the multi-drug resistance gene (MDR-1). Thecells are thus much less sensitive to drugs exported via P-glycoprotein(P-gp). See Harker, W. G. et al. Multi-drug (pleiotropic) resistance indoxorubicin-selected variants of the human sarcoma cell line MES-SA.Cancer Research, 1985, 45, 4091-4096. Similar growth inhibition curvesagainst the parent and resistant cell lines (FIGS. 8A and 8B) stronglysuggest that ORI 1313 and ORI 1327 are not substrates for the P-gpmulti-drug exporter. This will be particularly advantageous insituations involving target cells expressing MDR-1.

EXAMPLE V Cytotoxic Polyamine Analogues do not Alter Cellular PolyamineLevels

To determine the effects of treatment with polyamine analogues oncellular polyamine levels, intracellular polyamine levels were measuredby high performance liquid chromatography (HPLC) after 11 hr treatmentwith ORI 1313 (32 μM) or ORI 1327 (3 μM). These results showed nosignificant effects on the polyamine levels after drug treatment (seeFIG. 9). This observation is consistent with the previous observationsin the field. Moreover, measurement of SSAT activity in treated cellsshowed no induction of this enzyme (data not shown).

A follow-up experiment showed that co-treatment with a 50-fold excessconcentration of putrescine did not block the cytotoxic effects of ORI1313 or ORI 1327 treatment. Co-treatment with 1 mM DFMO did not alterthe results observed with ORI 1313 or ORI 1327 Co-treatment with apotent polyamine transport inhibitor, ORI 1202 (with K_(i) values of32±15 nM and 29±9 nM versus ³H-spermidine and ³H-putrescine uptake,respectively, in MDA cells and which effectively depletes cellularputrescine and spermidine when used in combination with a polyaminebiosynthesis inhibitor such as DFMO), also did not alter the observedinhibition of cell growth by ORI 1313 or ORI 1327.

The cellular uptake mechanism for these polyamine analogues was furtherexplored by their inhibition of ³H-putrescine uptake. Putrescine waspreviously shown to have a K_(m) of 12.4 μM for uptake into MDA cells.ORI 1313 showed a K_(iapp) of 28.3 μM versus putrescine uptake into MDAcells and 24.1 μM into PC-3 cells. ORI 1327 showed a K_(iapp) of 14.3 μMversus putrescine uptake into PC-3 cells. These results suggest that thepolyamine transporter is a possible mode of cellular uptake for theseanalogs. Nevertheless, since co-treatment with 50-fold excessputrescine, co-treatment with the polyamine biosynthesis inhibitor DFMO(previously shown to stimulate the uptake of polyamine analogs via thepolyamine transporter) and co-treatment with the potent polyaminetransport inhibitor ORI 1202 all failed to modify the effects of theanalogues, it appears that the analogues may enter cells by alternativeuptake mechanisms.

Since it has been established that tumor cells have a greaterrequirement for polyamines and this higher requirement is met by theincreased uptake and biosynthesis of polyamines (see Heston, W. D. W. etal. Differential effect of alpha-difluoromethylornithine on the in vivouptake of C-14 labeled polyamines and methylglyoxal bis(guanylhydrazone)by a rat prostate-derived tumor. Cancer Res. 1984, 44, 1034-1040;Minchin, R. F. et al. Paraquat is not accumulated in B16 tumor cells bythe polyamine transport-system. Life Sci., 1989, 45, 63-69; McCormack,S. A. et al. Putrescine uptake and release by colon cancer cells. Am. J.Physiol. 1989, 256, G868-G877; and Dave, C. et al. Studies in themechanism of cytotoxicity of methylglyoxal bis(guanylhydrazone) incultured leukemia L1210 cells. Adv. Polyamine Res. 1978, 1, 153-171),the polyamine analogue nature of ORI 1313 and ORI 1327 might cause themto be selectively accumulated in tumor cells. Byers and coworkers showedthat the uptake of a series of benzylated spermine analogs withanti-malarial properties was distinct from the polyamine transportsystem (see Byers, T. L. et al. Bis(benzyl)polyamine analogues aresubstrates for a mammalian cell transport system which is distinct fromthe polyamine-transport system. Biochem. J. 1990, 269,35-40).

EXAMPLE VI Involvement of Caspase-3 in Polyamine Analogue Cytotoxicity

Involvement of the pro-apoptotic caspase-3 enzyme in the action of OR1313 is shown by the experiment represented in FIG. 10. Briefly, PC-3cells were plated at 1000 cells/well in a 96-well plate and allowed toadhere for 1 day. 10 μM of ORI 1313 and 0, 5, or 15 μM Z-DEVD-fmk, acaspase inhibitor, were added and the cells were incubated for 3 days.Cell growth was evaluated by MTS assay (Promega). Vertical axisrepresents cell number as a % of no drug treatment controls.

Treatment with 10 μM ORI 1313 alone caused a 46% inhibition of growth ofPC-3 prostate cancer cells. When the cells were co-treated with 10 μMORI 1313 and 5 or 15 μM of the caspase inhibitor Z-DEVD-fmk the growthinhibition decreased to 19% and 10%, respectively. This indicates thatinhibition of a caspase enzyme reduced the cytotoxicity of ORI 1313,suggesting that such an enzyme plays a role in the cytotoxic mechanismof ORI 1313.

In a separate experiment, caspase-3 protease activity was measured inPC-3 cells treated with ORI 1313 (30 μM) and ORI 1327 (2 μM) for 14 hr.Doxorubicin (5 μM) was used as a positive control. The ApoAlertCaspase-3 Assay Kit from Clontech Laboratories was used to monitor theproteolytic activity. Untreated control cells were determined to have10.2±0.3 units/2×10⁶ cells of caspase-3 activity. Doxorubicin treatedcells showed 13.5±0.2 units/2×10⁶ cells of activity. ORI 1313 and ORI1327 showed 12.7±1.2 and 11.6±0.8 units/2×10⁶ cells of activity. Theseresults confirmed the activation of caspase-3 activity in PC-3 cellsafter treatment with ORI 1313 or ORI 1327. Alterations in polyamineanalogue concentration and treatment time may further increase caspaseactivity beyond that observed with doxorubicin.

Without being bound by theory, the polyamine analogues of the presentinvention may participate in an apoptosis mechanism by having an effecton mitochondria permeability transition and affecting the innermitochondrial membrane where the release of cytochrome c is induced.This is based on comparisons with recent reports on the effects ofpolyamines on mitochondria permeability transition. If a polyamineanalogue of the invention acts via such a mechanism, it would representa unique mechanism of action compared to other polyamine cytotoxicagents. Experimental confirmation of this mechanism is available by 1)measuring cellular uptake and localization of these agents using HPLCanalysis; 2) measuring the mitochondrial permeability transition onisolated mitochondria using light-scattering methods; 3) Western-blotanalysis of the release of cytochrome c from isolated mitochondria andwhole cells; and 4) exploring the sequence and timing of induction ofthe steps to apoptosis with these agents and compare to standardapoptosis-inducing agents. These experiments would be based uponwell-established literature procedures and would not require undueexperimentation.

EXAMPLE VII Selectivity for Melanoma Cells by Polyamine Analogues 1313and 1327

ORI 1313 and ORI 1327 were evaluated by the National Cancer Institute(NCI) with a 60-cell line screen. A surprising effectiveness wasobserved for ORI 1313 against 6 of 8 melanoma cell lines tested, whichis consistent with previous observations with N¹, N⁴-dibenzylputrescinein a melanoma xenograft animal model (see Frydman et al.) and suggests adramatic selectivity at least for melanoma cell lines. Comparison ofIC₅₀ values determined by the present inventors and NCI confirms thesedata: ORI 1313 in MDA cells, 12 vs. 2.3 μM, in PC-3 cells, 20 vs. 11.2μM; ORI 1327 in MDA cells, 0.65 vs. 0.005 μM, in PC-3 cells, 0.65 vs.0.81 μM.

EXAMPLE VIII Solubility and In Vivo Toxicity of Pol Amine Analogues

The dihydrochloride salt of ORI 1313 is soluble in 5% DMSO (dimethylsulfoxide) to at least 40 mM. ORI 1327 displays a more limited aqueoussolubility. The maximum solubility of the lactate salt of ORI 1327 is 5mM. The dihydrochloride salt is less soluble. Significant improvementsin the solubility were made by the use of hydroxypropyl-β-cyclodextrin(45% w/v HβC in H₂O). A 40 mM solution of ORI 1327 was produced in thismanner.

Mouse tolerance to drug treatment has been evaluated for ORI 1313 andORI 1327. Acute toxicities following single intraperitoneal (i.p.)injections of ORI 1313 or ORI 1327 were evaluated in BALB/c mice. ORI1313 was tolerated at 93 mg/kg. A dose of 186 mg/kg caused death in theanimals (mice appeared lethargic and had ruffled fur before death). ORI1327 was tolerated at 37 mg/kg, and a dose of 75 mg/kg caused death inthe animals. Chronic toxicities were evaluated by giving i.p. injectionsonce daily for 5 consecutive days. ORI 1313 and ORI 1327 were toleratedat 40 mg/kg and 7.5 mg/kg doses, respectively. Dosages of 80 and 30mg/kg, respectively, proved lethal. These data, in comparison with theforegoing in vitro experiments, indicate that potentially efficaciousdosages of these compounds are tolerated in mice. The approximatemaximum tolerated dosages in a one i.p. injection a day for 5 daysregimen are 40 mg/kg for ORI 1313 and 7.5 mg/kg for ORI 1327.

EXAMPLE IX Analogue Cytotoxicity against Human Tumor Xenografts in NudeMice

ORI was tested in a melanoma tumor xenograft tumor models in BALB/c nudemice. The A375 human melanoma cell line was chosen due to good activityby the analogue against this line in the NCI's 60-cell line test and thelow IC₅₀ value determined against this cell line (ORI 1313, 4.1 μM). Thexenograft efficacy study would be performed with two drugconcentrations. There were four groups of 10 mice. Treatment groupsreceived the maximum tolerated dose (MTD) or ½ MTD of each analogue.Compared to a negative control group treated with 5% dextrose, ORI 1313inhibited A375 melanoma growth: 36% growth inhibition at 25 days and 6.2day tumor growth delay (see FIG. 13). The drug was well tolerated up to7 weeks. Some transient skin ulceration and weight loss was seen after 7weeks of treatment. A positive control group treated with dacarbazineDTIC (dosage of 180 mg/kg i.p. once per day for 5 days) was used tovalidate this tumor model system.

Female BALB/c nu/nu athymic mice weighing 20 grams were the animal hostfor the human xenografts. The method of administration was i.p.injections with 9 mM or 4 mM ORI 1313 once daily for the duration of thestudy (end point when control tumors reached 2000 mg). The mode ofdelivery and dosing schedule may be further optimized by routinemethods. DITC was administered by i.p. injection once daily for 5 days.Treatment started when the tumors reach 5 mm×5 mm size (50 mg). Themouse weight and tumor size were measured twice weekly. The tumormeasurements by calipers were then converted to mg tumor volume by theformula L²×W/2. Once the control tumors reached 2000 mg both the controland treatment group mice were weighed, sacrificed and the tumorsremoved. The actual weights of tumors were then used to calculate thepercent growth inhibition by each analogue using the formula: % growthinhibition=(mean treated tumor weight/mean control tumorweight×100)−100.

All references cited herein are hereby incorporated by reference intheir entireties, whether previously specifically incorporated or not.As used herein, the terms “a”, “an”, and “any” are each intended toinclude both the singular and plural forms.

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

While this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth.

1. A polyamine analogue having the structure selected from the groupconsisting of:


2. The analogue according to claim 1, wherein said structure is:


3. The analogue according to claim 1 wherein said structure is:


4. The analogue according to claim 1 wherein said structure is:


5. A pharmaceutical composition useful for treating a disease orcondition in which the inhibition of cell growth or proliferation isdesirable, comprising a polyamine analogue according to claim 1, and apharmaceutically acceptable excipient.
 6. The composition of claim 5further comprising one or more additional agents known to be useful fortreating said disease or condition.
 7. A method for treating a diseaseor a condition in a subject associated with undesired cell growth orproliferation which comprises administering to said subject an effectiveamount of a polyamine analogue according to claim
 1. 8. The method ofclaim 7 wherein said undesired cell growth or proliferation isassociated with proliferation of cells of the immune system, cells ofthe vascular neointima, tumor cells or with undesired angiogenesis. 9.The method according to claim 7 wherein said disease or condition iscancer or post-angioplasty injury.
 10. The composition according toclaim 5 wherein said disease is osteoporosis.
 11. The composition ofclaim 5 formulated for intravenous, subcutaneous, intramuscular,intracranial, intraperitoneal, topical, oral, transdermal, intravaginal,intranasal, intrabronchial, intraocular, intraaural or rectaladministration.
 12. The composition of claim 5 formulated as anointment, cream, gel, solution, suspension, emulsion, powder, liniment,or salve.
 13. The method of claim 7 wherein said disease isosteoporosis.